SCIENTIFIC OPINION. EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 2,3

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1 EFSA Journal 2015;13(1):3966 SCIENTIFIC OPINION Scientific Opinion on the safety and efficacy of Biomin C3 (Bifidobacterium animalis ssp. animalis, Lactobacillus salivarius ssp. salivarius and Enterococcus faecium) as feed additive for chickens for fattening, chickens reared for laying and minor avian species other than laying species 1 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 2,3 ABSTRACT European Food Safety Authority (EFSA), Parma, Italy Biomin C3 is a preparation of strains of Enterococcus faecium, Bifidobacterium animalis and Lactobacillus salivarius. It is currently authorised in the European Union for use in feed for chickens for fattening. Application now has been made for an extension of use to chickens reared for laying and minor avian species other than laying species, for use in water for drinking and for the removal of a maximum dose. A tolerance study using water as the delivery system showed that consumption of 100 times the currently authorised maximum authorised dose in feed did not cause adverse effects in chickens for fattening. Thus, delivery of comparable doses of the additive via water for drinking is considered to be as safe for chickens for fattening as delivery via feed. Inclusion of a dose of colony-forming units (CFU)/L in the tolerance study without ill effects indicates that there would be no safety implications if the current maximum dose for chickens for fattening were withdrawn. The conclusions on safety for chickens for fattening, including the need for a maximum dose, would also apply to chickens reared for laying and minor avian species to the point of lay. Biomin C3 has the potential to improve the performance of chickens for fattening at a minimum dose of CFU/kg of complete feedingstuffs or the equivalent dose ( CFU/L) when delivered in water for drinking. The additive is also considered to have the potential to be efficacious when used in chickens reared for laying and minor avian species to the point of lay at a minimum dose of CFU/kg of complete feedingstuffs or CFU/L water for drinking. The additive is compatible with the coccidiostats robenidine hydrochloride, maduramycin ammonium, diclazuril, decoquinate, narasin, nicarbazin and narasin/nicarbazin. European Food Safety Authority, 2015 KEY WORDS zootechnical additives, gut flora stabilisers, Biomin C3, safety, efficacy, coccidiostats 1 On request from the European Commission, Question No EFSA-Q , adopted on 9 December Panel members: Gabriele Aquilina, Vasileios Bampidis, Maria De Lourdes Bastos, Lucio Guido Costa, Gerhard Flachowsky, Mikolaj Antoni Gralak, Christer Hogstrand, Lubomir Leng, Secundino López-Puente, Giovanna Martelli, Baltasar Mayo, Fernando Ramos, Derek Renshaw, Guido Rychen, Maria Saarela, Kristen Sejrsen, Patrick Van Beelen, Robert John Wallace and Johannes Westendorf. Correspondence: FEEDAP@efsa.europa.eu 3 Acknowledgement: The Panel wishes to thank the members of the Working Group on Micro-organisms, including Pier Sandro Cocconcelli, Andrew Chesson and Ingrid Halle, for the preparatory work on this scientific opinion. Suggested citation: EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed), Scientific Opinion on the safety and efficacy of Biomin C3 (Bifidobacterium animalis ssp. animalis, Lactobacillus salivarius ssp. salivarius and Enterococcus faecium) as feed additive for chickens for fattening, chickens reared for laying and minor avian species other than laying species. EFSA Journal 2015;13(1):3966, 10 pp. doi: /j.efsa Available online: European Food Safety Authority, 2015

2 SUMMARY Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of Biomin C3 when used in diets for chickens reared for laying and minor avian species other than laying species. The additive has been previously assessed for safety and efficacy when used in diets for chickens for fattening and is currently authorised in the European Union for this purpose at a minimum dose of colony-forming units (CFU)/kg and a maximum dose of CFU/kg of complete feedingstuffs. Application has been made for an extension of use to include chickens reared for laying and minor avian species other than laying species when added to their diets at the same minimum content as applied to chickens for fattening. In addition, the applicant is seeking authorisation for use in water for drinking for chickens for fattening and the additional species and the removal of the maximum dose from the present authorisation. A tolerance study using water as the delivery system showed that consumption of 100 times the currently authorised maximum dose in feed did not cause adverse effects in chickens for fattening and confirmed the conclusion on safety previously published. Thus, delivery of comparable doses of the additive via water for drinking is considered to be as safe for chickens for fattening as delivery via feed. The tolerance study included chickens for fattening exposed to a dose of CFU/L water without ill effects. Since it is not feasible to reach this dose in practice, any accidental or deliberate overdosing will always be below this value. Since this dose did not cause any detectable adverse effects, there seems to be no purpose in maintaining a maximum dose for chickens for fattening and there would be no safety implications if it were withdrawn. The conclusions on safety for chickens for fattening are taken to include chickens reared for laying. The FEEDAP Panel considers that safety for minor avian species to the point of lay can be assumed without the need for specific testing. In both cases, the conclusion on the need for a maximum dose would apply. The results of an additional performance study with chickens for fattening confirm the previous opinion that Biomin C3 has the potential to improve the performance of chickens for fattening at a minimum dose of CFU/kg of complete feedingstuffs and that the same conclusion applies when the additive is delivered at the equivalent dose ( CFU/L) in water for drinking. The additive is also considered to have the potential to be efficacious when used with chickens reared for laying and minor avian species to the point of lay at a minimum dose of CFU/kg of complete feedingstuffs or CFU/L water for drinking. The additive is compatible with the coccidiostats robenidine hydrochloride, maduramycin ammonium, diclazuril, decoquinate, narasin, nicarbazin and narasin/nicarbazin. EFSA Journal 2015;13(1):3966 2

3 TABLE OF CONTENTS Abstract... 1 Summary... 2 Background... 4 Terms of reference... 4 Assessment Introduction Additional information on characterisation Conditions of use Evaluation of the analytical methods by the European Union Reference Laboratory (EURL) 7 3. Safety for the target species Safety for chickens for fattening Conclusions on safety for chickens for fattening Safety for chickens for laying and for minor avian species to the point of laying Efficacy Efficacy for chickens for fattening Efficacy for chickens reared for laying and for minor avian species to the point of laying Compatibility with coccidiostats Post-market monitoring... 9 Conclusions... 9 Documentation provided to EFSA References EFSA Journal 2015;13(1):3966 3

4 BACKGROUND Regulation (EC) No 1831/ establishes the rules governing the Community authorisation of additives for use in animal nutrition. In particular, Article 4(1) of that Regulation lays down that any person seeking authorisation for a feed additive or for a new use of a feed additive shall submit an application in accordance with Article 7. In addition, Article 13(3) of that Regulation lays down that if the holder of an authorisation proposes changing the terms of the authorisation by submitting an application to the Commission, accompanied by the relevant data supporting the request for the change, the Authority shall transmit its opinion on the proposal to the Commission and the Member States. The European Commission received a request from Biomin GmbH 5 for authorisation of the product Biomin C3, Bifidobacterium animalis ssp. animalis (DSM 16284), Lactobacillus salivarius ssp. salivarius (DSM 16351) and Enterococcus faecium (DSM 21913), when used as a feed additive for chickens for fattening, chickens reared for laying and minor avian species other than laying species (category: zootechnical additives; functional group: gut flora stabilisers) under the conditions mentioned in Table 1. According to Article 7(1) of Regulation (EC) No 1831/2003, the Commission forwarded the application to the European Food Safety Authority (EFSA) as an application under Article 4(1) (authorisation of a feed additive or new use of a feed additive) and Article 13(3) (modification of the authorisation of a feed additive). EFSA received directly from the applicant the technical dossier in support of this application. 6 According to Article 8 of that Regulation, EFSA, after verifying the particulars and documents submitted by the applicant, shall undertake an assessment in order to determine whether the feed additive complies with the conditions laid down in Article 5. The particulars and documents in support of the application were considered valid by EFSA as of 21 May The additive Biomin C3 is a preparation of Bifidobacterium animalis ssp. animalis (DSM 16284), Lactobacillus salivarius ssp. salivarius (DSM 16351) and Enterococcus faecium (DSM 21913). This product is authorised for use in chickens for fattening. 7 EFSA issued an opinion on the safety and efficacy of Biomin C3 for chickens for fattening (EFSA FEEDAP Panel, 2012a). TERMS OF REFERENCE According to Article 8 of Regulation (EC) No 1831/2003, EFSA shall determine whether the feed additive complies with the conditions laid down in Article 5. EFSA shall deliver an opinion on the safety for the target animals, consumer, user and the environment and the efficacy of the product Biomin C3 (Bifidobacterium animalis ssp. animalis (DSM 16284), Lactobacillus salivarius ssp. salivarius (DSM 16351) and Enterococcus faecium (DSM 21913), when used under the conditions described in Table 1. 4 Regulation (EC) No 1831/2003 of the European Parliament and of the Council of 22 September 2003 on additives for use in animal nutrition. OJ L 268, , p Biomin GmbH, Industriestraβe 21, A-3130, Herzogenburg, Austria. 6 EFSA Dossier reference: FAD Commission Implementing Regulation (EU) No 544/2013 of 14 June 2013, concerning the authorisation of Bifidobacterium animalis ssp. animalis DSM 16284, Lactobacillus salivarius ssp. salivarius DSM and Enterococcus faecium DSM 21913, OJ L 163, , p. 13. EFSA Journal 2015;13(1):3966 4

5 Table 1: Description and conditions of use of the additive as proposed by the applicant Additive Registration number/ec No/No Category(-ies) of additive Functional group(s) of additive Biomin C3 4b1890 Zootechnical Gut flora stabilisers Composition, description Prespatation of Bifidobacterium animalis ssp. animalis (DSM 16284, CFU/g), Lactobacillus salivarius ssp. salivarius (DSM 16351, CFU/g) and Enterococcus faecium (DSM 21913, CFU/g) Trade name Name of the holder of authorisation Description Chemical Purity criteria formula Not applicable Biomin C3 Biomin GmbH Complies with EU law in microbial quality, heavy metals, toxins and undesirable substances Method of analysis Plate count following standard EN methods No (B. animalis), No (L. salivarius) and No (E. faecium) Species or category of animal chickens for fattening chickens reared for laying minor avian species other than laying species Maximum Age Slaughter age or start of laying Conditions of use Minimum content Maximum content Withdrawal period CFU/kg of complete feedingstuffs CFU/kg complete feedingstuff or CFU/L water for drinking none none Specific conditions or restrictions for use Specific conditions or restrictions for handling Post-market monitoring Specific conditions for use in complementary feedingstuffs Other provisions and additional requirements for the labelling Store in cool and dry place. R42, potential respiratory sensitizer Post-marketing monitoring will be carried out by Biomin GmbH in compliance with EU Regulations concerning Feed Hygiene and Feed and Food Controls, namely HACCP and Traceability, routine post-marketing sampling and analysis, and formal monitoring of customer feedback through product or service complaints none Maximum Residue Limit (MRL) Species or category of Target tissue(s) or Maximum content in Marker residue animal food products tissues EFSA Journal 2015;13(1):3966 5

6 ASSESSMENT 1. Introduction The additive Biomin C3 is a microbial preparation consisting of single strains of Bifidobacterium animalis ssp. animalis, Lactobacillus salivarius ssp. salivarius and Enterococcus faecium. The product should have a minimum content of CFU/g E. faecium, of CFU/g B. animalis ssp. animalis and CFU/g L. salivarius ssp. salivarius. Biomin C3 is currently authorised for use in feed for chickens for fattening at a minimum dose of CFU/kg and a maximum dose of CFU/kg of complete feedingstuffs. The applicant is now seeking an extension of the current authorisation to include chickens reared for laying and minor avian species other than laying species and the use of the additive in water for drinking for all these target species. In addition, the applicant proposes the removal of the current maximum recommended dose for chickens for fattening and the simultaneous use of the additive with coccidiostats. The safety of the additive for consumers, users and the environment has been assessed in the previous opinion and did not give rise to concerns (EFSA FEEDAP Panel, 2012a). The safety for chickens for fattening and the efficacy of the product when used in broiler feed was also established at that time. The FEEDAP Panel is unaware of any new data which would lead it to revise its conclusions. Therefore, the focus of the present opinion is on the consequences of the new use in water for drinking, for the additional target animals and the proposed removal of a maximum dose. 2. Additional information on characterisation The additive which is the subject of the present application has the same formulation and method of manufacture as that considered in the previous application for chickens for fattening (EFSA FEEDAP Panel, 2012a). Thus, the data pertaining to impurities, physical properties and shelf life still apply. The data on stability in premixtures, mash and pelleted feed for chickens for fattening are considered applicable to chickens reared for laying and for minor avian species, given the similarity in feed formulation. Since delivery of the additive via water for drinking was not proposed in the application for use with chickens for fattening, stability in water was tested. 8 Three batches of the additive were suspended in tap water at CFU/L, the minimum concentration proposed. Individual counts were made of the three component strains at the start of the experiment and after 24 and 48 hours of incubation at 30 C. Essentially no losses were observed after 48 hours and the relative proportions of the three strains remained unchanged. The battery of antibiotics currently recommended for the testing of antibiotic resistance amongst lactic acid bacteria were, with a single exception, all included in the recommended list applicable at the time of the submission made for chickens for fattening. 9 The results presented in the application for chickens for fattening are still valid and the conclusion on the susceptibility of the strains to all tested antibiotics remains unchanged. A new requirement for the testing of enterococcal strains against tylosin was introduced in the latest revision of the guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance (EFSA FEEDAP Panel, 2012b). E. faecium DSM was tested and found to be susceptible Conditions of use The additive is intended for use with chickens reared for laying and minor avian species other than laying species at the minimum dose of CFU/kg feed currently authorised for chickens for fattening. An equivalent dose of CFU/L when the additive is delivered via water for drinking 8 Technical dossier/section II/Annex II Technical dossier/section II/Annex II Technical dossier/section II/Annex II.36. EFSA Journal 2015;13(1):3966 6

7 is proposed. No maximum dose is proposed for the new target species and the applicant is seeking removal of the present maximum dose applied when used in feed for chickens for fattening Evaluation of the analytical methods by the European Union Reference Laboratory (EURL) The EURL considered that the conclusions and recommendations reached in the previous assessment are valid and applicable for the current application Safety for the target species 3.1. Safety for chickens for fattening On the basis of a tolerance study in which chickens for fattening showed no adverse effects when given a dose of CFU/kg feed of the additive, the FEEDAP Panel concluded that Biomin C3 is safe for chickens for fattening at doses up to CFU/kg of complete feedingstuffs, the currently authorised maximum dose (EFSA FEEDAP Panel, 2012a). 12 A new tolerance study was carried out using water for drinking as the delivery vehicle. 13 The study was designed to provide evidence in support of the removal of the maximum content. A total of 480 birds (Ross 308), of mixed sex, were allocated to one of three treatment groups with eight replicates of 20 birds per treatment. All birds were given the same mash diet for 35 days. Treatments consisted of: A control group provided with water for drinking without addition of the additive. A group given water for drinking containing CFU/L. This dose was selected to be equivalent to the highest dose given in the previous tolerance study, in which the additive was delivered via feed ( CFU/kg feed), and corresponds to a 100-fold overdose the currently maximum authorised dose in feed. A group given water for drinking containing CFU/L. This density could be obtained only by using a concentrated cell mass of the organisms constituting the additive, rather than the additive itself, and represents a fold overdose of the equivalent maximum authorised dose in feed. Treated water was freshly prepared daily and any water remaining from the previous day was removed and discarded. Samples of the treated water (week 1 and week 4) were analysed for microbial counts, including differential counts, to confirm the intended dose. Animals health status was monitored daily and body weight was measured after 1, 14 and 35 days. Feed intake was measured on a pen basis and used, together with the average body weight gain per pen, to calculate the average feed to gain ratio. All data were analysed for normal distribution (Kolmogorov Smirnov test). This was followed by a Levene test for homogeneity of variances. If the Levene test was non-significant, it was followed by an analysis of variance (ANOVA) with a post-hoc Bonferroni test to distinguish between the single groups. If significant differences were found in the Levene test, a Welch test with a post-hoc Tamhane test was used. Data that were not normally distributed were analysed using a non-parametric approach (Kruskal Wallis). Overall mortality was low (< 2.0 %) and was not treatment related. No health issues requiring intervention were noted. There was a significant increase in final body weight in the 100 overdose group compared with the control and overdose groups (1.89 vs vs kg), and this was reflected in the figures for average daily gain. However, the control group and the high dose group did not significantly differ. Feed intake was essentially constant across groups and the calculated feed to gain ratio (1.96 vs vs. 1.91) did not significantly differ, although there was a numerical improvement seen in the 100 group, again reflecting the higher final body weight. 11 The full report is available on the EURL website: 12 Technical dossier/section III/Annexes III Technical dossier/section III/Annexes III EFSA Journal 2015;13(1):3966 7

8 3.2. Conclusions on safety for chickens for fattening Biomin C3 for poultry A tolerance study using water as the delivery system showed that consumption of 100 times the currently authorised maximum dose did not cause adverse effects in chickens for fattening and confirmed the conclusion on target animal safety previously published. The FEEDAP Panel in its statement on the use of additives in water for drinking established the principle that safety was determined not by the route of delivery (water or feed) but by the extent of exposure (EFSA FEEDAP Panel, 2010). The result of the tolerance studies demonstrate that, where doses are matched, the same results are to be expected. Consequently, delivery of the additive via water for drinking is considered to be as safe for chickens for fattening as delivery via feed. It is not physically possible to reach a dose of CFU/L, or its equivalent in feed, using the additive and so any accidental or deliberate overdosing will always be below this value. Since this dose did not cause any detectable adverse effects, there seems to be no purpose in maintaining a maximum dose for chickens for fattening and there would be no safety implications if it were withdrawn Safety for chickens for laying and for minor avian species to the point of laying The conclusions on safety for chickens for fattening are taken to include chickens reared for laying. As safety for a relevant major species (chickens for fattening) has been established, the FEEDAP Panel considers that safety for minor avian species to the point of lay can be assumed without the need for specific testing. The conclusion on the need for a maximum dose would also apply. 4. Efficacy 4.1. Efficacy for chickens for fattening In the previous opinion on chickens for fattening, the results of five trials were considered (EFSA FEEDAP Panel, 2012a). Final body weight was significantly improved in two of the five trials and feed to gain ratio in another two. Data from the trials were tested for homogeneity and pooled to enable a statistical meta-analysis. The results showed a significant increase in final body weight (1.73 kg vs kg, P < 0.05) but no significant improvement in feed to gain ratio due to supplementation with Biomin C3 was observed. On this basis it was concluded that the additive had the potential to increase final body weight when used in feed at a minimum dose of CFU/kg complete feed. An additional study now has been provided to demonstrate that the same production benefit is to be expected when the additive is supplied via water for drinking. 14 A total of 320 Ross 308 chicks, of mixed sex, were randomly allocated to one of two groups. The first group was provided with water for drinking without supplementation and the second group with water for drinking containing CFU/L of the additive (dose confirmed by analysis including differential counts from samples taken during week 1 and week 4). Water for both groups was provided ad libitum, but that for the treated group was prepared freshly each day. Each group was housed to provide eight replicates of 20 birds/replicate. Both groups received the same mash diet without the additive. The duration of the study was 35 days. Birds were monitored for signs of illhealth throughout the study and body weight and feed intake were recorded. Average feed intake and body weight/pen was used to calculate the feed to gain ratio. Data were checked for normal distribution (Kolmogorov Smirnov test). If normally distributed, an independent-samples t-test with included F-test (Levene test) for homogeneity of variances was carried out; if not, a non-parametric Kruskal Wallis test was applied. Overall mortality was low (~ 1.6 %) and no health problems requiring intervention were observed. The final body weight in the treated group was significantly higher than in the control group (1.89 vs. 14 Technical dossier/section III/Annexes IV EFSA Journal 2015;13(1):3966 8

9 1.99 kg, P = 0.009). This difference was also reflected in the average daily gain and total weight gain data. Feed intake and feed to gain ratio were not significantly different between groups, although there was, as would be expected given the final body weights, a numerical improvement in feed to gain ratio (1.96 vs. 1.87). The results of this study with chickens for fattening confirm the previous opinion that Biomin C3 has the potential to improve the performance of chickens for fattening at a minimum dose of CFU/kg of complete feedingstuffs and that the same conclusion applies when the additive is delivered at the equivalent dose ( CFU/L) in water for drinking Efficacy for chickens reared for laying and for minor avian species to the point of laying The conclusion on chickens for fattening also applies to chickens reared for laying. Since efficacy has been demonstrated when the additive is used with chickens for fattening and, as it is reasonable to assume that the mechanism of action would be the same in other growing avian species, the Panel does not see a need for specific studies. Consequently, the additive is also assumed to have the potential to be efficacious when used in minor avian species to the point of lay at a minimum dose of CFU/kg of complete feedingstuffs or CFU/L water for drinking. 5. Compatibility with coccidiostats For all three component strains of the additive, minimum inhibitory concentration values were at least four times greater than the maximum authorised concentration of robenidine hydrochloride, maduramycin ammonium, diclazuril, decoquinate, narasin, nicarbazin and narasin/nicarbazin 15 Therefore, compatibility with these coccidiostats is assumed. 6. Post-market monitoring The FEEDAP Panel considers that there is no need for specific requirements for a post-market monitoring plan other than those established in the Feed Hygiene Regulation 16 and Good Manufacturing Practice. CONCLUSIONS A tolerance study using water as the delivery system showed that consumption of 100 times the currently authorised maximum dose did not cause adverse effects in chickens for fattening and confirmed the conclusion on safety previously published. Delivery of comparable doses of the additive via water for drinking is considered as safe for chickens for fattening as delivery via feed. It is not physically possible to reach a dose of CFU/L, or its equivalent in feed, using the additive and so any accidental or deliberate overdosing will always be below this value. Since this dose did not cause any detectable adverse effects, there seems to be no purpose in maintaining a maximum dose for chickens for fattening and there would be no safety implications if it were withdrawn. The conclusions on safety for chickens for fattening are taken to apply also to chickens reared for laying. The FEEDAP Panel considers that safety for minor avian species to the point of lay can be assumed without the need for specific testing. In both cases the conclusion on the need for a maximum dose would apply. The results of the additional study with chickens for fattening confirm the previous opinion that Biomin C3 has the potential to improve the performance of chickens for fattening at a minimum dose 15 Technical dossier/section II/Annexes II.57 and Regulation (EC) No 183/2005 of the European Parliament and of the Council of 12 January 2005 laying down requirements for feed hygiene. OJ L 35, , p. 1. EFSA Journal 2015;13(1):3966 9

10 of CFU/kg of complete feedingstuffs and that the same conclusion applies when the additive is delivered at the equivalent dose ( CFU/L) in water for drinking. The additive is also considered to have the potential to be efficacious when used with chickens reared for laying and minor avian species to the point of lay at a minimum dose of CFU/kg of complete feedingstuffs or CFU/L water for drinking. The additive is compatible with the coccidiostats robenidine hydrochloride, maduramycin ammonium, diclazuril, decoquinate, narasin, nicarbazin and narasin/nicarbazin. DOCUMENTATION PROVIDED TO EFSA 1. Biomin C3. Zootechnical feed additive for chicken for fattening, chickens reared for laying and minor poultry species other than laying species. March Submitted by Biomin GmbH. 2. Comments from Member States received through the ScienceNet. REFERENCES EFSA Panel on Additives and Products or Substances Used in Animal Feed (FEEDAP), Scientific Opinion on the use of feed additives authorised/applied for use in feed when supplied via water. EFSA Journal 2010;8(12): pp. doi: /j.efsa EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2012a. Scientific Opinion on the safety and efficacy of Biomin C3 (Enterococcus faecium, Bifidobacterium animalis and Lactobacillus salivarius) for chickens for fattening. EFSA Journal 2012;10(12): pp. doi: /j.efsa EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2012b. Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance. EFSA Journal 2012;10(6): pp. doi: /j.efsa EFSA Journal 2015;13(1):